This readme.txt file was generated on 2023-02-16 by Mor Ben-Tov. The files included in this dataset include the data required to reproduce the figures and analyses included in Ben-Tov et al., “A neural hub for holistic courtship displays”. A preprint version of this article can be found at: https://doi.org/10.1101/2021.09.09.459618 GENERAL INFORMATION 1. Title of Dataset: Data from: A neural hub for holistic courtship displays 2. Author Information A. First Author Contact Information Name: Mor Ben-Tov Institution: Duke University OCRID ID: 0000-0003-0283-1724 Email: morbentov@gmail.com B. Corresponding Author Contact Information Name: Richard Mooney Institution: Duke University OCRID ID: 0000-0002-3308-1367 Email: mooney@neuro.duke.edu C. Additional Author Contact Information Name: Fabiola Duarte Institution: Duke University ORCID ID: 0000-0001-6147-7978 Email: fabiola.duarteortiz@duke.edu 3. Date of data collection (single date, range, approximate date): 2018-2022 4. Geographic location of data collection: Durham, NC, United States of America SHARING/ACCESS INFORMATION 1. Licenses/restrictions placed on the data: Data are available under a CC-BY license. DATA & FILE OVERVIEW 1. Folder overview: This dataset has 9 folders, 7 of them are named after the figure for which the data was used. The Source Data folder contains quantification data for additional figures. The Code folder contains four subfolders with Matlab scripts to analyze behavior (Behavior Analysis), calcium imaging (Fiber Photometry), and song (Motif Count and Syllable Notes Analysis) data. Three of these subfolders also contain raw sample data to run the scripts. Additional raw data can be provided by the First Author upon request. We have deposited 7 types of data in the repository: (1) Confocal microscope images of A11 inputs and output tracing (corresponding to Figure 1 and Figure S1) (2) Confocal microscope images of in situ hybridization (Figure 2 and Figure S5) (3) Audio recordings of undirected songs and introductory notes used to determine the spectral properties before and after 6-OHDA lesions (Figures S2 and S3) (4) Fiber photometry signals aligned to the first motif or the first introductory note (Figure 6) (5) Source data (Figures 3-5, S2 and S5). (6) Matlab code to perform behavioral, fiber photometry, and song analysis.(7) Raw sample data in the form of .avi, .wav, .mat or .csv files to run the analysis code. METHODOLOGICAL INFORMATION 1. Description of methods used for collection/generation of data: Tissue collection: Birds were deeply anesthetized with intramuscular injection of 20 µl Euthasol (Virbac), and transcardially perfused with 0.025 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were removed, post-fixed in 4% PFA at 4°C overnight and moved to cryoprotective 30% sucrose PFA solution for two days. Frozen sagittal sections (thickness of 50 or 75µm) were prepared with a sledge microtome (Reichert) and collected in PBS. Immunofluorescence: Floating tissue sections were washed three times in PBS, permeabilized with 0.3% Triton X-100 in PBS (PBST) for 10 minutes, blocked in 10% blocking reagent for 1 hour (Nacalai Tesque, 06349-64), and incubated with either primary antibody against TH, GFP, mCherry, or synapsin (1:1000, AB152; Millipore/Sigma; A11120, Invitrogen; ab167453, Abcam; 106 011, Synaptic Systems) at 4°C overnight. Sections were then washed three times in PBS and incubated with a fluorophore conjugated secondary antibodies (1:500; Invitrogen, A-21245, A21207, A11001) in PBS at room temperature for 2-4h, followed by three washes in PB. Sections were coverslipped with Fluoromount-G (SouthernBiotech), and then imaged with a confocal microscope (Zeiss) through a 20x or 10x objective lens controlled by Zen software (Zeiss). To label A11 projections and inputs, AAV2/9.CAG-scGFP (made in Duke’s Viral Vector Core), dextrans (Alexa Fluor 488, D-22910, ThermoFisher) or retrobeads (LumaFluor) were injected into the A11 of adult male birds. Incubation times before perfusion were 4 weeks for viral expression and 4–7 days for tracers. Images were processed with ImageJ to adjust for brightness and contrast. For the analysis of TH+ neurons in A11 after 6-OHDA treatments, neurons with diameter greater than 10 µm were counted manually. Floating section in situ hybridization chain reaction (HCR): Birds were perfused with 4% PFA/PBS and post-fixed in the same solution overnight and then in 30% sucrose in RNAse-free PBS for 2 overnights at 4°C. Brains were then sectioned at 75 µm and collected into 0.5-1% PFA (4% PFA diluted in RNAse-free PBS). At room temperature, slices were first washed twice in PBS for 3 min, incubated in 5% SDS/PBS for 45 min, rinsed twice with 2x sodium chloride sodium citrate 0.1% Tween 20 (2x SSCT), and put in 2x SSCT for 15 min on a shaker. Then, they were pre-incubated in probe hybridization buffer for 30 min at 37°C, and later hybridized in 2.5 µL probe set/500 µl probe hybridization buffer overnight at the same temperature. The probes were custom made by Molecular Instruments to detect zebra finch isoforms of VGluT2 (Vesicular glutamate Transporter 2) (NCBI Reference Sequence: NM_001309508.1), TH (Tyrosine hydroxylase) (XM_002198931.3), and VGAT (Vesicular GABA transporter) (XM_002189664.2). The next day, slices were washed four times for 15 min with 500 µL of probe wash buffer at 37°C and twice in 2x SSCT for 5 min at room temperature on a shaker. Then they were incubated in 500 µl of HCR amplification buffer for 30 min at room temperature on a shaker. Last, slices were incubated in a solution containing 300 µL HCR amplification buffer and fluorescent hairpins for the HCR initiator probe for 2 overnights, in the dark at 25°C. On the last day, at room temperature, slices were washed twice with 2x SSCT for 5 min, stained with Neurotrace for 2 hours (1:500, N21479; Invitrogen), rinsed twice with 2x SSCT and mounted on a slide with Fluoromount-G. Hairpins, probe sets and probe hybridization buffer were created by Molecular Instruments. HCR for TH and VGluT2 was performed on sections from 4 birds, HCR for TH and VGAT was performed on sections from 2 birds, and HCR for TH, VGluT2 and VGAT was performed on sections from 1 bird. Lesion experiments: A pair of male and female adult zebra finches were housed in an isolated soundproof box. The male and the female were separated by electronic glass (HOHOFILM Electronic PDLC, Smart Film) that was connected to an external switch. When powered off, this glass is opaque, preventing the male and the female from seeing each other; they could hear each other in either condition. In order to record female-directed singing, the experimenter powered the glass on, rendering it transparent and enabling the birds to see each other. The use of electronic glass eliminated the need to handle the birds, increasing the probability that the males would sing to the female. Video recordings started approximately 20 seconds prior to visibility onset. The glass remained transparent for 1-7 minutes. We recorded female-directed singing 2-5 times a day, allowing enough time between exposures to let the main return to baseline levels of arousal (at least 40 minutes between exposures; with a mean interval of 97±54 minutes). Video recordings stopped approximately 20 seconds after visibility offset. Baseline singing rates were recorded for 5-7 days, after which birds were divided into 4 experimental groups, HVC 6-OHDA, HVC sham, A11 6-OHDA (6-hydroxydopamine), and A11 sham. Videos of the birds were recorded using webcams (Genius WideCam F100). Songs were automatically recorded with Sound Analysis Pro (SAP2011 (Tchernichovski et al., 2000)). Singing rates were calculated manually by counting all female-directed songs produced during the first minute of female presentation and by counting all undirected songs produced during a 4-hour period each day. Female-directed and undirected song rates were calculated for five days prior to surgery. An average singing rate over this baseline period was calculated and then used to normalize singing rates for each day pre- and post-surgery. Injection of 6-OHDA: Adult male birds received bilateral injections of either 400nl 6-OHDA solution into HVC or 80-100nl 6-OHDA solution into A11 (N=4 for A11 and N=5 HVC). The solution was PBS-based and included 10-60mM 6-OHDA hydrochloride (Tocris, 2547), 10 µM l-ascorbic acid (Millipore/Sigma, A92902), and 1 µM desipramine hydrochloride (Tocris, 3067), which was included as an inhibitor for noradrenaline and serotonin transporters to protect noradrenergic and serotonergic neuron terminals at the injection site. Control birds received an injection of PBS with 10 µM ascorbic acid and 1 µM desipramine (N=6 for A11 sham group and N=5 for HVC sham group). Drugs were dissolved in PBS immediately before injection in place of equimolar NaCl (working solution: ~300 mOsm, pH 7.3). After injection, birds were returned to their original home cage until approximately 14 days post injection. Fiber photometry imaging: Adult male birds were injected with pAAV-hSynapsin1-axon-GCaMP6s-P2A-mRuby3 (axon-target GCaMP6s) bilaterally into A11 or HVC. After waiting a minimum of 3 weeks for viral expression, birds were anesthetized and placed in a stereotaxic apparatus. Bilateral craniotomies were made over HVC and fiber optic ferrules (200 um core, 0.37 NA, Neurophotometrics) were implanted. For all recordings, axon-targeted GCaMP6s was excited at two wavelengths (470nm for imaging of calcium-dependent signals and 415nm for an interleaved isosbestic control to eliminate motion artifacts). An sCMOS camera was used to capture fluorescence (FP3001, Neurophotometrics) at 30 Hz. Synchronized video and sound recordings were acquired using a webcam (Logitech). Data acquisition was performed with custom Bonsai code and data analyzed using custom-written Matlab scripts. In each imaging session, the signal from the isosbestic control channel was first smoothed and then regressed to the signal from the calcium-dependent channel. To calculate the calcium-dependent signal, first the linear model generated from regression was used to generate a predicted control signal. Then the calcium-dependent signal was calculated by subtracting the predicted control signal from the raw calcium-dependent signal (Figure S5F). Audio recordings were filtered using a third-order median filter. Introductory notes, syllables and different types of calls were labeled manually in Matlab. Calcium signals were aligned to the audio recordings and then z-scored to normalized changes in fluorescence across animals. Onset times were defined as the time for which the signal reached 5% from its peak value. Hybridization chain reaction for identification of Fos expression:In order to minimize off-target Fos mRNA detection, birds were perfused 30 minutes after cage lights first turned on in the morning. For female-directed singing (n = 4 birds), 4-5 females were presented sequentially to maximize motif amounts. Live video was monitored to ensure no significant undirected (facing away from the female, disengaged) singing occurred during female presentations. For the undirected condition (n = 4 birds), birds were allowed to sing freely in the morning for the 30-minute window. Lights were then turned off and birds were immediately perfused. Brains were processed for HCR as described in a previous section, the custom probes designed by Molecular Instruments targeted Fos (XM_002200534.5), TH and VGAT. For each bird, a z-stack encompassing A11 was collected at 40x power to accurately visualize Fos signal, along with a TH channel. All image processing was done with ImageJ. First, the TH and Fos channels were noise-subtracted (20 µm rolling ball radius), and TH channel was smoothed, automatically thresholded (otsu method), and converted to a binarized mask. The mask was then transferred to the Fos channel, which was then also thresholded. Fos particles within the TH mask were then quantified for intensity (expressed as a fraction of the TH mask). For any additional details see preprint or contact the authors. 2. Methods for processing the data: Song analysis: Vocalizations of >5 ms were detected by thresholding of the recorded sounds. Pairwise similarity was quantified as percentage of similarity (asymmetrical similarity) between song motif and/or introductory notes of different experimental groups using SAP2011 (Tchernichovski et al., 2000) with default parameters for zebra finches, and reported as similarity score. The percentage of similarity was calculated for representative song motifs and introductory notes randomly chosen from treated birds, before and after 6-OHDA treatment, and averaged across all comparisons. Individual syllables and introductory notes were determined using custom MATLAB graphical user interfaces (Tumer & Brainard, 2007) to further measure their duration and inter-note gap duration. Video analysis: Videos were analyzed for 20 seconds prior to female presentation and 30 seconds after female presentation. For each video, the bird’s position and head orientation were measured using either DeepLabCut (3/9 birds, Mathis et al., 2018) or custom MATLAB graphical user interfaces (6/9 birds, M. Ben-Tov) that enable the marking of the bird’s body position and head orientation across video frames.Two experimenters scored the videos (the scores given by the two experimenters were not significantly different (paired t-test, p=0.54). For each video, the position was normalized to the cage size, to allow a comparison between different videos and birds. For any additional details see preprint or contact the authors. 3. Instrument- or software-specific information needed to interpret the data: Confocal microscope image files: ZEN (Zeiss) and/or ImageJ (NIH) Audio files: Windows Media Player (Microsoft) or Quicktime (Apple) Matlab codes: Matlab R2020b (Mathworks) Video files:Windows Media Player (Microsoft) or Quicktime (Apple) Fiber photometry files: Excel (Microsoft) DATA-SPECIFIC INFORMATION FOR: Data files for Figure 1 and Figure S1 are named according to the relevent figure panels. Data files for 6-OHDA lesion data (Figure S2, S3) were divided to two subfolders: A11 and HVC. Within each folder, additional subfolders with the birds' ID are included. Each individual file is labelled with "pre" or "post" to indicate the vocalizations prior or after the lesion. The HVC folder includes an extra folder IN (introductory notes), which contains song files used to carry out the introductory note analysis on Figure S3. Data for fiberphotometry data is labeled with A11 or HVC at the begining, indicating whether the signal comes from A11 axons in HVC or local HVC axons. Also in the file name an indication to which element in the vocalization the data were aligned to: first motif or first introductory note. Source data Data for TH+ Fos+ cell count is labeled to indicate whether the bird was singing female directed songs or undirected songs. Columns indicate number of cells that were eithere TH+, Fos+ or both. Data for Singing Rates includes columns with data for each bird, experimental day, area treated, context (directed-dir or undirected- undir), number of motifs quantified, and the normalized number of motifs sang. This file is missing normalized singing rates for A11 and HVC undirected controls/sham lesioned birds. Data for IN and Calls includes columns with details on experimental conditions and quantification of average introductory notes and female-directed calls when the female was visible. Data for Looking at Female includes columns with data identifiers of experimental birds and conditions, and the fraction of total time the male spent looking at the female obtained from the video analysis. Data for Movement Analysis is the same as Looking at Female but includes the total percentage of displacement towards (positive values) or away from (negative values) the female. Data for TH GCaMP area indicates the quantification of either TH+ area or GCaMP and TH+ area (obtained by the same method described for Fos expression) in HVC for four different slices for each experimental bird. Data for A11 cell count includes number of TH+ cells that were quantified in either A11 or VTA.